球孢白僵菌热激蛋白的生物学功能解析及其调控真菌耐热力的分子机理

Heat shock proteins (HSPs) are a family of intracellular products induced rapidly under heat stress conditions and highly diverse in molecular size and function. Apart from the basic function of their protecting cells from heat damage, HSPs act as chaperones to affect peptide folding and protein configuration, which may cause significantly more profound biological reactions in cells than cellular thermotolerance. For this reason, much attention has been paid to the basic and applied aspects of HSPs in model and human-pathogenic fungi worldwide in the past few years but no effort has been made to investigate the HSPs of entomopathogenic fungi such as Beauveria bassiana, a classic fungus used in mycoinsecticides. Since fungal thermotolerance is of special importance for the field persistence and adaptation of fungal formulations to high temperatures during summer, we propose to initiate an intensive study on various HSP members of B. bassiana by means of our multi-stress response experimental systems of insect pathogens established in the past few years and collaborative achievements in comparative genomic analysis of several fungal entomopathogens. Our objectives are: (1) to elucidate the biological functions of individual HSPs in B. bassiana genome by constructing knockout or knockdown mutants and characterizing their phenotypic parameters in association with the transcripts of other affected genes and possible mechanisms involved in the regulation of the functions, (2) to find possible HSP genes that are responsible for specific tolerance to heat and thus potential for use in genetic engineering of fungal candidates for improved field persistence and efficacy of mycoinsecticides, and (3) to discover fungal HSP members with novel functions that are exploitable in crop breeding and medicine development.

热激蛋白(Hsp)是一类高温胁迫诱导下生物体内迅速产生的生理反应产物，种类多，生物学功能因成员不同而异，除了具有增强细胞耐受高温胁迫能力的基本功能之外，还充当分子伴侣角色而影响肽链折叠及相关蛋白构型，由此诱导超越热激反应范围、意义更加深远的一系列生物学反应，成为近年来国际上的研究热点，但罕有昆虫病原真菌的相关研究报道。本项目旨在以球孢白僵菌为主要研究对象，基于本实验室建立起来的生防真菌多胁迫反应分析系统和不同抗逆性状的定量评价技术以及比较基因组学的合作研究成果，运用正向遗传操作技术构建各类Hsp基因的敲除株/回补株或沉默株，结合应用蛋白组学、代谢组学手段，深入研究和揭示该菌不同Hsp成员的主效生物学功能及其表达调控模式，一是找到Hsp家族中可能存在的特异性耐热响应成员，用于真菌杀虫剂田间适应性和稳定性的遗传改良；二是发掘具有新颖生物学功能的热激蛋白基因资源，用于农作物改良和生物医药开发。

热激蛋白Hsp大家族是真核细胞在高温、辐射、氧化等环境胁迫下表达并发挥特定功能的一类分子伴侣。本课题以球孢白僵菌的Hsp家族为研究对像，试图通过解析这一蛋白大家族主要成员的生物学功能及表达调控模式，揭示主要成员对球孢白僵菌适应昆虫寄主及其环境的意义，明确它们对球孢白僵菌生物防治潜能的贡献。球孢白僵菌有三个具HSF结构域的转录因子(Hsf1、Hsf2/Sfl1及Hsf3/Skn7)，参与调控30个Hsp基因的转录表达。运用农杆菌介导的真菌转化体系构建各Hsp及Hsf的单基因缺失株和回补株，共成功敲除和回补21个基因在，而Hsp40、Hsp70及Hsp90家族分别有2、9和2个基因是敲除致死的。Hsf1和17个Hsp敲除株耐45℃高温胁迫能力显著低于野生株，其中Hsp98、Hsp78、Hsp40a及Hsf1失活后分生孢子失活一半所需的时间缩短50%以上，说明它们热激应答中扮演关键角色。UV-B紫外辐射使Hsf3和15个Hsp敲除株分生孢子失活一半的辐射剂量显著低于野生株，其中Hsp98失活后降幅达50%。除Hsp70e以外，20个敲除株的毒力都显著低于野生株。在生长胁迫实验中，所有敲除株对甲萘醌或双氧水的氧化胁迫更为敏感，抑制50%生长所需的浓度(EC50)显著低于野生株。NaCl高渗胁迫抑制11个Hsp和3个Hsf敲除株的EC50值都低于野生株，尤其Hsp40a敲除株对高渗胁迫极为敏感。胞壁干扰胁迫剂刚果红对21个敲除株的生长抑制作用都大于野生株，其中5个低分子Hsp、Hsf1和Hsf2敲除株的EC50不及野生株的一半。这些结果表明，大多数Hsp蛋白及Hsf转录因子在球孢白僵菌生防潜能的维持中起着重要程度不同的作用。选择表型变化突出的敲除株进行多方面的深入研究，发现在进化中获得的具有细菌源DnaJ结构域、分子量分别仅为46.86 (Mas5)和56.68 kD (Mdj1)的两个Hsp40家族成员是其重要代表。两者参与调控生长、产孢、孢子萌发、孢壁组装、胞内Na+平衡、多胁迫应答、昆虫体壁降解酶分泌、寄主侵染等一系列细胞过程关联蛋白的转录表达和后修饰，因而是维持白僵菌生防潜能所必不可少的。因此，Mas5、Mdj1等小分子Hsp基因具有潜在利用价值，可用于球孢白僵菌生物防治潜能的遗传改良。本项目还研究解析了与Hsp家族关联的若干信号和效应蛋白基因的功能及表达调控模式。